Tissue contraction is an integral part of wound closure; however, excessive or abnormal contraction of tissues can lead to pathological contractures, tissue deformation and loss of tissue function, all major health problems in the United States. The long-term goal of this project is to understand the cellular basis underlying wound contraction and tissue contracture by studying the formation and function of the myofibroblast. Myofibroblasts are specialized fibroblasts that express smooth muscle alpha-actin (SMAA) and assemble contractile structural elements as part of their ability to generate the contractile force responsible for tissue contraction. The mechanical properties of the extracellular matrix are critical in regulating SMAA expression, as well as other SM-specific cytoskeletal proteins, and myofibroblast formation and function. These results have led to a series of novel hypothesis regarding mechano-regulation of myofibroblast formation and function: (i) mechano-regulation of the SM-specific cytoskeletal expression in myofibroblasts is mediated by changes in actin dynamics; (ii) myocardin-related transcription factor A (MRTF-A) couples mechano-regulated changes in actin dynamics with gene expression by translocating from cytoplasmic actin to the nucleus; (iii) MRTF-A activates a subset of SM-specific cytoskeletal proteins, in addition to SMAA, in myofibroblasts; (iv) MRTF-A activation of this subset of SM-specific cytoskeletal proteins is responsible for the formation and function of myofibroblasts. We will test these hypotheses by use of tissue culture and animal wound models in which response to different mechanical environments can be examined. In addition, we will knock down expression of MRTF-A and SMAA using siRNA to determine their role in myofibroblast formation and function. Relevance to Public Health: In order to control wound healing and the devastating effects of pathological contractures it is essential to regulate the formation and function of the myofibroblast. This proposal will test whether myofibroblast formation and function can be regulated by targeting specific signaling pathways and transcriptional regulatory events and the effect this has on wound healing and tissue contracture in animal models. This study will provide the basis for translating basic science findings on the myofibroblast formation and function into the development of novel therapeutic approaches to control wound healing and pathologic contractures.